JPH0139439B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is directed to the new antibiotic neoviridoglysein.
For more information on Cl and its production method, see the formula Antibiotic neoviridoglysein-Cl-
c and the antibiotic neoviridoglysein-Cl-t
(c and t each represent a stereoisomer containing amino acid residues derived from cis-chloroproline and trans-chloroproline) and their production methods. The present inventors previously proposed the following general formula (In the formula, R ₁ represents a methyl group or an ethyl group,
R ₂ represents a hydrogen atom, a hydroxyl group or a methyl group. ) Antibiotic neoviridoglysein (a compound in which R ₁ is an ethyl group and R ₂ is a hydrogen atom),
Same (compounds where R ₁ is a methyl group and R ₂ is a hydrogen atom), Same (compounds where R ₁ is an ethyl group and R ₂ is a hydroxyl group), Same (compounds where R ₁ is a methyl group and R ₂ is a hydroxyl group) ) and the same MP (R ₁ is a methyl group and R ₂
represents a methyl group) and methods for producing them (see, for example, Japanese Patent Publication No. 55-3339 and Japanese Patent Application Laid-open No. 57-64654). The present inventors conducted research to find a more useful analogue of the antibiotic neoviridoglyzein (hereinafter simply referred to as "neoviridoglyzein"), and as a result, discovered that a known neoviridoglyzein-producing bacterium By adding 4-chloroproline or a substance containing 4-chloroproline to the nutrient medium during the culture of Completed the invention. Neoviridoglyzein-Cl-c and neoviridoglysein-Cl-t provided by the present invention
are stereoisomers, respectively, as described below,
All of them exhibit approximately the same antibacterial activity as the neoviridoglyseins represented by the general formula (), and are useful as pharmaceuticals, veterinary drugs, or feed additives. Another invention provided by this application is the formula ()
Neoviridoglysein-Cl-c and/
Alternatively, it is a method for producing neoviridoglysein-Cl-t. The method for producing the antibiotic includes culturing a microorganism belonging to the genus Streptomyces that has the ability to produce the antibiotic in a known nutrient medium by adding 4-chloroproline or a substance containing 4-chloroproline; This can be carried out by collecting the antibiotic from the culture. The microorganisms used in the practice of the present invention can be microorganisms belonging to any genus as long as they have the ability to produce the above-mentioned antibiotics.
Preferably, Streptomyces Sp. Examples include strains deficient in prolyl hydroxylase activity obtained by mutation of the strain. In addition, among the mutant strains, specific ones include:
On September 20, 1981, the applicant submitted the No. 184 Microbiological Research Institute to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
Streptomyces Sp.G-89 deposited as No.
Bacterial strains can be mentioned. The mycological properties of this strain are as follows. The strain used in this application extends short colorless aerial hyphae from well-branched, simple-branched basal hyphae, and forms a loose spiral spore chain at the tip. The surface of the spore is smooth. No whorl or ascospore formation is observed. The culture characteristics of this strain in various media are as follows. Growth on sucrose/nitrate agar medium: Slight growth Aerial hyphae: Thin white aerial hyphae formed here and there Substrate hyphae: Colorless to grayish-white Soluble pigment: None Growth on glycol/asparagine agar medium: Good Aerial hyphae: Most No formation, but white if formed Substrate hyphae: Pale yellow-white to light yellow (2db-2fb) Soluble pigment: None Growth on glycerin/asparagine agar medium: Moderate Aerial hyphae: Hardly formed, barely formed White basal hyphae: pale yellow to grayish yellow (2db~2dc~3ec) Soluble pigment: none Growth on yeast malt agar medium: good Aerial hyphae: white to slightly grayish white Baseline hyphae: bright yellow (2fb ), but becomes brown-gray (3li) in the later stages of cultivation. Soluble pigment: absent or sometimes slightly brown. Growth on starch agar medium: Moderate Aerial hyphae: Rarely formed, but when formed, white basal hyphae: pale yellow (2fb), center of colony exhibits light grayish yellowish brown (3ge). Soluble pigment: None Starch decomposition: Slightly separated Growth on tyrosine agar medium: Moderate Aerial hyphae: Hardly formed, but white aerial hyphae are occasionally observed. Base hyphae: Gray-yellow (3ec) to light gray-yellowish brown (3ge) Soluble pigment: At the early stage of culture, it produces light purple to light reddish-brown pigment, but after about 10 days of culture, it becomes light brown.
It then becomes thinner (a small amount of melanin pigment is produced). Growth on nutrient agar medium: Good aerial hyphae: Forms thin white aerial hyphae. Base hyphae: pale yellow (2db) Soluble pigment: none Growth on oatmeal agar medium: good Aerial hyphae: white to grayish-white Baseline hyphae: gray-yellow (3ec) to light gray reddish-brown (4ge) Soluble pigment: none This strain The optimum temperature range for growth is 25-33°C, but it can also grow at 20°C or 37°C, although with poor growth. However, it cannot grow above 52℃. This strain liquefies gelatin on a glucose-peptone-gelatin medium and hydrolyzes starch, albeit weakly, on an inorganic salt-starch agar medium. This strain peptonizes skim milk. However, no coagulation was observed. In peptone yeast iron agar medium and tryptone yeast liquid medium, this strain
It does not produce melanin-like pigments, but it sometimes does in tyrosine agar media. This strain uses D-xylose, D-glucose, D-fructose, L-rhamnose, and D-mannite, but does not use L-arabinose, i-initol, or raffinose, and uses sucrose on Pridham-Gottlieb agar medium. use slightly. The strain used in this application is morphologically classified as S (Spirales).
It belongs to L. section, and grows white to light grayish-white aerial mycelia on yeast malt agar medium.
Do not use arabinose. This strain suppresses the production of neoviridoglyzein (pyridoglyzein), which is the main product of neoviridoglyzein by normal manufacturing methods,
It is possible to increase the selectivity in the production of neoviridoglysein-Cl-c and neoviridoglysein-Cl-t according to the present invention, and is more suitable for achieving the objects of the present invention. Next, a method for producing the antibiotic of the present invention using the above-mentioned producing bacteria will be described. To produce the above neoviridoglysein-Cl-c and neoviridoglysein-Cl-t,
The above strain is inoculated into a nutrient medium suitable for the production of the antibiotic, and the culture medium is heated at 20°C to 33°C, preferably at 24°C to 33°C.
The product is aerobically cultured at ℃ for 2 to 14 days, and extracted and purified from the culture. As the medium components, known ones used for culturing strains belonging to the genus Streptomyces can be used. Add a suitable carbon source, such as soluble starch, glucose, and sucrose, a nitrogen source, such as soybean flour, cottonseed meal, dried yeast, etc., and an inorganic salt, such as dibasic potassium phosphate, salt, etc., to a medium at an appropriate concentration, e.g. 0.1mM or more,
Preferably, 0.5 to 8mM of 4-chloroproline or a substance containing 4-chloroproline is added for culturing. Cultivation is carried out under aerobic conditions at 20°C to 33°C, preferably at 24°C.
Carry out the culture at ~30℃ and keep the pH at 5.5~ during culture.
It is recommended to adjust it to 8.0. Neoviridoglysein-Cl accumulated in the culture medium
-c, -Cl-t and neoviridoglyseins containing it can be isolated by known methods for isolating depsipeptide antibiotics and various purification methods based on the physical properties of the substance. Furthermore, if necessary, it can be recovered as a mixture of neoviridoglyseins. For example, neoviridoglyseins containing neoviridoglysein-Cl-c and neoviridoglysein-Cl-t present in the culture solution are benzene, toluene, ethyl acetate,
It is extracted with water-repellent organic solvents such as butyl acetate, n-butanol, methylene chloride, chloroform, and methyl isobutyl ketone. In addition to extraction methods using organic solvents, activated carbon and Amberlite XAD
(Manufactured by Rohm and Haas) etc., gel filtration with Cephadex LH-20 (Manufactured by Pharmacia) etc., adsorption chromatography with alumina or silica gel, etc. can be used in appropriate combinations using column methods or thin layer methods. can. In some cases, a countercurrent distribution method using an appropriate solvent can also be used for recovery and purification. Next, the physicochemical properties of neoviridoglysein-Cl-c and neoviridoglysein-Cl-t of the present invention will be described.
Neoviridoglysein-Cl-c and neoviridoglysein-Cl-t are methanol, ethanol, propanol, n-butanol, dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride, chloroform. , carbon tetrachloride, N,N-
It is a white amorphous solid that is soluble in dimethylformamide, dimethyl sulfoxide, and diethyl ether, sparingly soluble in water and isopropyl ether, and insoluble in petroleum ether and hexane. Neoviridoglysein-Cl-c and neoviridoglysein-Cl-t
is stable for one month at a temperature of 37°C or less in an aqueous solution at a pH of 9.0 or less. In addition, PH2.5 when treated at 60℃ for 30 minutes
It is stable in the range of ~8.0. The ultraviolet absorption maximum measured in methanol and the E ¹ % ₁ cm value at that wavelength (inside the box) are shown below. Neoviridoglyzein - Cl - c305nm (90) Neoviridoglyzein - Cl - t305nm (90) Ultraviolet absorption maximum in 0.1N methanolic caustic soda solution and E ¹ % ₁ cm value at that wavelength (inside the cutlet) is shown below. Neoviridoglysein-Cl-c340nm (88) Neoviridoglysein-Cl-t340nm (86) The molecular weight determined by mass spectrometry is shown below. Neoviridoglyzein-Cl-c896 Neoviridoglyzein-Cl-t896 In mass spectrometry, a molecular ion peak appeared at 896, and an intensity peak about 1/3 of the molecular in-peak was observed at 898. It is supported that each of the antibiotics has one chlorine atom in each molecule. The Rf values obtained by thin layer chromatography developed using the ascending method using silica gel plate 60F254 manufactured by Merck are shown below. 1 Solvent system 1 (chloroform: methanol = 30:
In the case of 1) Neoviridoglyzein-Cl-c Rf0.47 Neoviridoglyzein-Cl-t Rf0.51 Neoviridoglysein (control) Rf0.41 2 Solvent system 2 (benzene: ethyl acetate: methanol : Acetic acid: Water = 50:50:10:10:3) Neoviridoglyzein-Cl-cRf0.62 Neoviridoglysein-Cl-tRf0.65 Neoviridoglysein (control) Rf0.60 Neoviridoglysein-Cl-c and neoviridoglysein-Cl-t
was hydrolyzed in 6N hydrochloric acid at 110°C for 16 hours, concentrated to dryness to thoroughly remove the hydrochloric acid, and subjected to thin layer chromatography (using Avicel Cellulose SF manufactured by Funakoshi Pharmaceutical Co., Ltd.).
(Developed using a solvent system of butanol:acetic acid:water = 4:1:1 or methanol:pyridine:water = 80:3:20), high-pressure filter paper electrophoresis (Toyo Roshi No.
51A, manufactured by Toyo Roshi Co., Ltd. Formic acid: Acetic acid: Water = 25:
60 min at 0°C using 75:900 PH1.8 buffer.
60V/cm) and amino acid analyzer (Hitachi)
835-50 type), amino acid analysis was performed.
The following amino groups and organic acids were detected. Neoviridoglysein-Cl-c: threonine,
Leucine, 4-chloroproline, sarcosine, β,N-dimethylleucine, alanine, phenylsarcosine, 3-hydroxypicolinic acid Neopyridoglysein-Cl-t: Threonine:
Leucine, 4-chloroproline, sarcosine, β,N-dimethylleucine, alanine, phenylsarcosine, 3-hydroxypicolinic acid Among these, 3-hydroxypicolinic acid was analyzed using thin layer chromatography (Merck silica gel plate 60F254). The Rf value is 0.46 when developed with chloroform:methanol = 2:1, and the Rf value when developed with n-butanol:acetic acid:water = 4:1:1 using Avicel Cellulose SF Funakoshi Pharmaceutical.
(0.60, which matched the Rf value of the standard sample) and its presence was confirmed by mass spectrometry. Thus, it was confirmed that 3-hydroxypicolinic acid exists as a constituent amino acid in neoviridoglyzein-Cl-c and neoviridoglyzein-Cl-t. To determine the stereoisomerism of 4-chloroproline,
After separating each 4-chloroproline, it was converted to N-acetyl methyl ester and subjected to gas chromatography (column: 3 mm x 1.5 m, carrier: Chromosolve W, stationary phase: 0.5% PEGA, carrier gas: N ₂ 1.7 Kg/cm ² , temperature: 178°C).
Next, the retention time is shown. Neoviridoglyzein-4-chloroproline in Cl-c 3.63 minutes Neoviridoglyzein-4-chloroproline in Cl-t 2.75 minutes Cis-4-chloroproline (standard material) 3.65 minutes Trans-4-chloroproline (standard material)
2.75 minutes Combining the above results, neoviridoglysein
The chemical structures of Cl-c and neoviridoglysein-Cl-t were determined to be the above formula (). Next, the antibacterial activity of the antibiotic against various pathogenic bacteria was demonstrated by comparing it with the known substance neoviridoglyzein (pyridoglyzein),
We will clarify its usefulness. Neoviridoglysein-Cl-c and neoviridoglysein-Cl-t
The following table also shows the minimum inhibitory concentrations against various microorganisms obtained using the same agar dilution method.

[Table] Also, neoviridoglysein-Cl-c and the same-
Cl-t shows a synergistic effect with griseoviridein as well as other neoviridoglyzeins. Neoviridoglyzein-Cl-c, neoviridoglysein-Cl-t, and griseoviridein alone have a concentration that does not show an inhibition zone and are applied to a paper disk at a constant concentration on an agar medium inoculated with the test bacteria. This is clear from the fact that when the disks are placed at a distance of 1 and cultured overnight, no inhibition circle is observed around each disk, but an inhibition circle is formed between the two disks. Therefore, although the new antibiotics neoviridoglyzein-Cl-c and neoviridoglyzein-Cl-t of the present invention are effective antibiotics alone, they are even more effective when mixed with griseoviridein. Becomes an effective antibiotic. In practice, it can be used as a mixture of neoviridoglyzeins and griseoviridein without separating each substance. Next, examples of the present invention will be given to illustrate the present invention more specifically. Example 1 50 ml of a medium consisting of 2.0% soluble starch, 0.2% yeast extract, 0.2% malt extract, and 0.1% soybean flour (adjusted to pH 6.8) was placed in a 250 ml Erlenmeyer flask, and after sterilization Streptomyces Sp. 89 (Microtechnology Research Institute No. 184
No.) was inoculated and cultured with rotary shaking at 28°C for 72 hours, and this was used as a seed mother. Soluble starch 2.0%, L
-Asparagine 0.5%, salt 0.1%, dibasic potassium phosphate 0.1%, magnesium sulfate 0.05%, zinc sulfate 0.01%, manganese chloride 0.001% (adjusted to PH 6.8)
After sterilization, 20 ml of the medium consisting of the following was placed in a 100 ml Erlenmeyer flask, and 0.2 ml of the seed mother was inoculated, followed by rotary shaking culture at 28° C. for 24 hours. Separately sterilized transformer-4
-Chloro-L-proline or cis-4-chloro-
L-proline was added to each flask at concentrations of 0.5, 1.0, 20, and 4.0 mM, and rotary shaking culture was continued for an additional 120 hours. After the cultivation was completed, 5 ml of the culture solution was extracted with 1 ml of benzene, and a certain amount of the extracted solution was spotted on a silica gel plate 60F254 manufactured by Merck. Manufactured by Shimadzu Corporation after developing with chloroform:methanol=30:1
When quantified using TLC-Chromatoscanner CS-910, the values shown in the table below were obtained.

[Table] Example 2 50 ml of a medium consisting of 2.0% soluble starch, 0.2% yeast extract, 0.2% malt extract, and 0.1% soybean flour (adjusted to pH 6.8) was placed in a 250 ml Erlenmeyer flask, and after sterilization Streptomyces Sp. G-89 (same as above) was inoculated and cultured with rotary shaking at 28°C for 3 days, and this was used as a seed mother. Soluble starch 2.0%, L-asparagine 0.5%, salt 0.1%, dibasic potassium phosphate 0.1%,
Medium 50 consisting of 0.05% magnesium sulfate, 0.01% zinc sulfate, and 0.001% manganese chloride (adjusted to PH 6.8)
After sterilization, 1 ml of the above seed mother was inoculated into a 250 ml Erlenmeyer flask and cultured with rotary shaking at 28°C. After 24 hours, sterilized trans-4-chloro-L-proline was added to a concentration of 4 mM, and the culture was continued. After culturing, culture for 6 days and filter the culture solution.48
ml of filtrate was obtained. After adjusting the pH of the filtrate to 7.5, it was extracted twice with 20 ml of ethyl acetate and concentrated to dryness. that
After dissolving in 2.5 ml of benzene, it was filtered to remove insoluble matter. Add benzene solution to Merck's silica gel
The samples were spotted on a 60F254 thin layer plate and thin layer chromatography was performed for adjustment. Development was carried out using chloroform:methanol=30:1. 3650 after deployment
Neoviridoglysein-Cl was irradiated with ultraviolet light of Å
- I scraped off part c. The scraped silica gel was extracted with 10 ml of methanol and dried. Further, the dried product was dissolved in 5 ml of methylene chloride, insoluble matter was removed by filtration, and then dried to obtain 2.2 mg of a single specimen of neoviridoglysein-Cl-c. Example 3 In the same manner as in Example 2, cis-4-chloro-L
- Cultured with the addition of proline. Using the recovery and purification method described in Example 2, a single standard of neoviridoglysein-Cl-t was obtained from the culture solution after 6 days of culture (850mcg).
I got it.

Claims (1)

[Claims] 1 formula Antibiotic neoviridoglysein-Cl-
c and the antibiotic neoviridoglysein-Cl-t
(c and t each indicate a stereoisomer containing amino acid residues derived from cis-chloroproline and trans-chloroproline). 2. A microorganism belonging to the genus Streptomyces and capable of producing the antibiotic neoviridoglysein-Cl-c and the antibiotic neoviridoglysein-Cl-t is cultured in a nutrient medium, and from the culture, the antibiotic A method for producing the antibiotic, which comprises collecting a substance. 3. A microorganism that belongs to the genus Streptomyces and has the ability to produce the antibiotic neoviridoglyzein-Cl-c and the antibiotic neoviridoglyzein-Cl-t is Streptomyces Sp. No. 3562) or Streptomyces Sp.G-
89 (Feikoken Joyori No. 184), the manufacturing method according to claim 2.